A major finding from the comprehensive sequencing efforts of cancer genomes is that nearly half of newly discovered driver genes whose mutations contribute to the tumor's initiation and progression encode proteins that directly regulate chromatin through modification of histones or DNA. This is best exemplified by four enzymes that are involved in gene silencing. Two families of enzymes, DNA methyltransferases and TET dioxygenases, catalyze the addition and removal of 5-methylcytosine, respectively. The other two, polycomb repressive complex 1 and 2 (PRC1 and PRC2), are functionally linked and catalyze histone H2AK119 monoubiquitylation and H3K27 trimethylation, respectively. Genes encoding for multiple subunits of these four enzyme complexes are found to be frequently mutated in different types of cancers. This investigation is aimed at determining three questions related to the epigenetic control by the ubiquitylation. (1) How are TET enzymes regulated by monoubiquitylation (Aim 1)? (2) Is H2AK119 monoubiquitylation also catalyzed by a PRC1-independent E3 ligase (Aim 2)? And (3) how is the silencing of the p15INK4B-p14ARF-p16INK4A gene cluster by DNA methylation and polycomb coordinated by the KDM2B/FBXL10 E3 ligase (Aim 3)? The investigation of these three questions is proposed based on four recent discoveries we made. (1) CRL4VprBP catalyzes monoubiquitylation on TET proteins to promote TET binding to chromatin, and this regulation is disrupted by multiple recurrent mutations in TET2 in leukemia. (2) TET enzymes are recruited to target genes by DNA sequence-specific transcription factor WT1. (3) Anti-obesity protein WDTC1 assembles a CRL4WDTC1 E3 ligase to catalyze H2AK119 monoubiquitylation and control adipogenesis. And (4) Histone demethylase KDM2B/FBLX10, which was recently shown to recognize unmethylated CpG and recruit PRC1, assembles an active E3 ubiquitin ligase. These findings led us to propose a focused investigation to determine epigenetic control by ubiquitylation, an area that, despite being the first to identify the substrate of ubiquitylation, remains poorly understood mechanistically and underappreciated functionally. This investigation uniquely combines our extensive experiences in the study of the p15INK4b-p14ARF-p16INK4a gene cluster, the ubiquitin pathway and epigenetic control. If accomplished, this project will reveal new mechanisms of epigenetic control by ubiquitylation; gain new insights into the regulation of one of the most frequently altered regions in human diseases, the p15INK4b-p14ARF-p16INK4a cluster, and help to better understand the crosstalk between two major mechanisms of gene silencing by DNA methylation and polycomb. As this investigation covers multiple enzymes, it also has the potential to identify new targets for therapeutic intervention.